Rescue of pyrimidine-defective Pseudomonas aeruginosa through metabolic complementation

ABSTRACT Chronic infections harbor multiple pathogens where dynamic interactions between members of the polymicrobial community play a major role in determining the infection outcome. For example, in a nutrient-rich polymicrobial infection, bacteria have the potential to undergo evolutionary changes that impair their ability to synthesize essential metabolites. This adaptation may facilitate metabolic interdependencies between neighboring pathogens and lead to difficult-to-treat chronic infections. Our research group previously demonstrated that Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA), typically considered classical competitors, can adopt a cooperative lifestyle through bi-directional purine exchange medicated by exogenous DNA (eDNA) release. To further validate our initial findings, in this study, we investigated the potential exchange of pyrimidine between PA and other pathogens, which is another constituent of DNA. In our findings, we observed that a pyrimidine-deficient transposon mutant strain of PA showed improved growth when co-cultured with wild-type PA, SA, Acinetobacter baumannii (AB), and Enterococcus faecalis (EF). Additionally, improved fitness of pyrimidine-deficient PA was further observed in chemical complementation with eDNA and uridine-5′-monophosphate. Interestingly, the rescue of PA growth through eDNA complementation is not as effective as in intact cells, such as SA, AB, EF, and wild-type PA, implying that eDNA is a lesser contributor to this metabolic complementation. Also, the exchange mechanism between pathogens involves more active mechanisms beyond simple eDNA or metabolite release. Our data further highlights the importance of cell-to-cell contact for effective and increased metabolic complementation. IMPORTANCE This research holds crucial implications for combating chronic infections, where multiple pathogens coexist and interact within the same environment. By uncovering the dynamic exchange of pyrimidines between Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA), our study reveals a previously unrecognized aspect of interspecies cooperation. The observed enhanced growth of a pyrimidine-deficient PA strain when co-cultured with SA suggests potential avenues for understanding and disrupting bacterial metabolic interdependencies in chronic infection settings. Furthermore, our findings highlight the mechanisms involved in metabolic exchange, emphasizing the importance of cell-to-cell contact. This research explored essential metabolic interactions to address the challenges posed by difficult-to-treat chronic infections.


Revision Guidelines
To submit your modified manuscript, log into the submission site at https://spectrum.msubmit.net/cgi-bin/main.plex.Go to Author Tasks and click the appropriate manuscript title to begin.The information you entered when you first submitted the paper will be displayed; update this as necessary.Note the following requirements: • Upload point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file • Upload a clean .DOC/.DOCX version of the revised manuscript and remove the previous version • Each figure must be uploaded as a separate, editable, high-resolution file (TIFF or EPS preferred), and any multipanel figures must be assembled into one file • Any supplemental material intended for posting by ASM should be uploaded separate from the main manuscript; you can combine all supplemental material into one file (preferred) or split it into a maximum of 10 files, with all associated legends included For complete guidelines on revision requirements, see our Submission and Review Process webpage.Submission of a paper that does not conform to guidelines may delay acceptance of your manuscript.
Data availability: ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide Spectrum production staff with the correct URL for the record.If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication may be delayed; please contact production staff (Spectrum@asmusa.org)immediately with the expected release date.
Publication Fees: For information on publication fees and which article types are subject to charges, visit our website.If your manuscript is accepted for publication and any fees apply, you will be contacted separately about payment during the production process; please follow the instructions in that e-mail.Arrangements for payment must be made before your article is published.
ASM Membership: Corresponding authors may join or renew ASM membership to obtain discounts on publication fees.Need to upgrade your membership level?Please contact Customer Service at Service@asmusa.org.
The ASM Journals program strives for constant improvement in our submission and publication process.Please tell us how we can improve your experience by taking this quick Author Survey.
Thank you for submitting your paper to Spectrum.

Sincerely, Justin Kaspar Editor Microbiology Spectrum
Reviewer #1 (Comments for the Author): This manuscript explores an initial observation that the growth of Pseudomonas aeruginosa pyrimidine auxotrophic mutants could be rescued by co-culture with Staphylococcus aureus.The authors measured eDNA in these co-cultures, attempted to rescue growth by supplementation with exogenous DNA, and measured bacterial growth when direct contact was prevented.Although the initial observation is interesting, the authors' conclusions that secreted eDNA mediates the growth rescue are not supported by their data.While eDNA was detected in the co-cultures, supplementation with exogenous DNA only caused a slight increase in growth of the pyrimidine mutants.There is also an overall lack of rigor in data interpretation and strain validation.

Specific comments:
Were the pyrimidine auxotrophs validated by growth in minimal medium with and without pyrimidine supplementation?Can the mutants be complemented?Does co-culture of pyrimidine mutants with wild-type PA14 restore growth?Is this response specific to S. aureus co-culture?
Ln 182 -'less virulent' does not seem like an appropriate term here, since these experiments were all performed in the absence of host cells Ln 194-196 -It's not clear how JE2 having more eDNA than the PA mutants validates the PA mutants' auxotrophy.The conclusion about DNA secretion vs cell lysis is also not clear.
Ln 210-211 -These two figures show that co-culture with JE2 can rescue growth of PA14 pyrimidine auxotrophs and that eDNA can be detected to varying extents in these cultures.eDNA as the cause of the growth rescue has not been established.
Fig 3 and 4 -These data seem to argue against the conclusion that eDNA mediates rescue of the pyrimidine auxotrophs.Very small growth differences are observed with eDNA supplementation and growth is not restored to the level of  does not indicate the difference between panels A and B. Without this information, it is impossible to interpret the results.Both panels show co-cultures between pyrB mutant and JE2, but they show different results.
Simplifying the abbreviations for S. aureus and P. aeruginosa by using just one abbreviation each (either SA/PA or the strain names JE2/PA14) would help the reader follow the manuscript.
Reviewer #2 (Comments for the Author): In this manuscript, Al Mahmud et al., present evidence that pyrimidines produced by S. aureus (SA) can rescue P. aeruginosa (PA) that lacks the ability to perform pyrimidine synthesis.The authors use a series of PA mutants created using a transposon each lacking a unique gene in pyrimidine synthesis.They then show that, alone, these mutant strains survive, but do not grow, in minimal medium.However, complementation with SA or herring DNA returns growth to the mutants.The authors then show that there is a likely a spatial component to the exchange.Taken together, the manuscript adds to our understanding of how PA and SA interact.
Overall, the manuscript is well written.The figures and figure legends are very clear, which I really appreciate.However, I am concerned that that it more than just pyrimidines that are involved in the rescue.It could be an entirely different substrate that feeds into pyrimidine synthesis that is overproduced by SA.I think that to truly show that it is pyrimidines that are recuing PA when co-culture with SA, the authors need to perform some important control experiments.

Specific points:
1) The introduction is really short and misses a lot of interactions that have been previously shown between these two pathogens, both antagonistic and mutualistic.These should be summarized.Furthermore, because this manuscript involves several genes and (presumably) nitrogenous base scavenging, a brief paragraph on purine/pyrimidine synthesis and salvage is warranted to put the mutants (pyrF, etc.) in the greater context of the pathways in which they are found.
2) Throughout the first part of the results and discuss, the reader is made to think that eDNA is being secreted by and that is rescuing PA.But SA could also produce metabolites that feed into pyrimidine synthesis, which may be rescuing PA.If it was simply a matter of providing pyrimidines, then shouldn't all of the mutants be rescued more or less in the exact same way.That is, in figure 3, shouldn't all of the mutants behave, for example, like pyrF?I understand that this data is not in the presence of SA but because cytosine enters pyrimidine synthesis after purF (and thus all of the other enzymes that they have knocked out) and thymine enters via a route that doesn't involve the pyr enzymes, shouldn't they all behave the same?This to me is very puzzling...If it was simply just pyrimidines, could the PA mutants be rescued with supplementation with pyrimidines?Have the authors tried these experiments (as opposed to herring DNA)?To me, that would provide more evidence that only pyrimidines are responsible for rescue.
3) Another way to provide support for pyrimidines in rescue in acquiring SA that lacks a gene in pyrimidine synthesis early on in the pathway (right after the prpp autoregulation edge in the pathway).Co-culture with this strain should not lead to growth of either strain.That would provide more conclusive evidence that it is pyrimidines (nitrogenous bases or the deoxy form) that is rescuing PA. 4) Can PA import/salvage deoxy nucleotides?Or are only nitrogenous bases imported?5) The authors use the dye picogreen to bind to eDNA.Based on their methods, I think that the picogreen assay was performed in the presence of intact (and maybe live) cells.How can the authors rule out that picogreen is not binding to DNA internal the cells.If I were to approach his problem, I would filter the medium to remove cells (with e.g., 0.45 um filter, which should remove cells but leave eDNA) and then stain the cell free medium.To me, this would ensure that this DNA is outside of the cells.6) Will cell free but SA conditioned medium rescue PA?If it does, then I think that the authors can certainly strengthen their claim that something secreted is rescuing PA.
Overall, I think that that authors have a really good idea, and if it is supported by additional, critical control experiments above, would certainly have an impact on the field.But without the experiments above, while it could be pyrimidines that are exchanged, it might also be a number of other things that are rescuing the PA mutant strains.
We really appreciate the reviewer's for their valuable feedback, comments and suggestions.We tried to address all the concerns raised by the reviewer's.We believe their feedback increased the quality of this manuscript.In the response section, the text highlighted in green is presented here as a reference to the direct intext revision in the clean manuscript.Thanks so much!Sincerely, Hafij Al Mahmud

Reviewer #1 (Comments for the Author):
This manuscript explores an initial observation that the growth of Pseudomonas aeruginosa pyrimidine auxotrophic mutants could be rescued by co-culture with Staphylococcus aureus.The authors measured eDNA in these co-cultures, attempted to rescue growth by supplementation with exogenous DNA, and measured bacterial growth when direct contact was prevented.Although the initial observation is interesting, the authors' conclusions that secreted eDNA mediates the growth rescue are not supported by their data.While eDNA was detected in the co-cultures, supplementation with exogenous DNA only caused a slight increase in growth of the pyrimidine mutants.There is also an overall lack of rigor in data interpretation and strain validation.Response: Thanks for highlighting this issue.Based on the reviewer's comment, we revisited our data, and also, based on the new additional data, we agree with the reviewer's concern that eDNA would be a minor contributor to pyrimidine complementation.We have revised the whole manuscript accordingly, including our concluding remarks in lines 380-387 "Both metabolically active and lysed cells may facilitate the pyrimidine complementation in neighboring pathogens.Importantly, secreted eDNA is a minor contributor in pyrimidine complementation towards rescuing the pyrimidine-defective PA, but neighboring cells (SA) are more proficient for pyrimidine exchange via intricate cell-to-cell contact mechanism.Apart from eDNA, secreted nucleotides, such as UMP, a precursor for pyrimidine nucleotide, may also rescue pyrimidine defective mutants.The role of secreted nucleosides and nitrogenous bases in this complementation requires further investigation."Also, we have validated one of the mutant strains, pyrB::tn, through whole genome sequencing (Please find the edits in line 115 and 263).

Specific comments:
Were the pyrimidine auxotrophs validated by growth in minimal medium with and without pyrimidine supplementation?Can the mutants be complemented?
Response: We really appreciate you pointing out this critical issue.Yes, the pyrimidinedefective mutants are defective in growth in RPMI (+1% casamino acid) minimal medium compared to wild-type PA14 (pyr::tn monoculture growth in Figures 1, 2, 4).According to your comment and suggestion, in addition to eDNA, we conducted a chemical complementation experiment using UMP, a precursor for pyrimidine nucleotide synthesis and a product of de novo pyrimidine biosynthesis.We found that UMP can also significantly rescue the growth of a pyrimidine-defective mutant of PA.Please find the added data in lines 261-263 and Supplemental Figure 1 "Furthermore, we confirmed the pyrB::tn mutant to be truly deficient in pyrimidine biosynthesis using chemical complementation with uridine-5′-monophosphate (Supplemental Figure 1) as well as whole genome sequencing (data not shown)" Because of time limitations, we could not include all the pyrimidine-defective mutants for the extended experiments.
Does co-culture of pyrimidine mutants with wild-type PA14 restore growth?Is this response specific to S. aureus co-culture?Response: To address your feedback, we have conducted a co-culture experiment using pyrB::tn as a representative pyrimidine-defective mutant with wild-type PA14, Enterococcus feacalis, and Acinetobacter baumannii.Our data showed that the growth of pyrB::tn can be rescued with any of these organisms, which means the rescue is not specific to SA. Please see the results in lines 256-273 and Figures 2A and B "Further, we wanted to explore whether the observed metabolic complementation of pyrimidine-defective PA is restricted to SA only or not.To this extent, we conducted similar experiments using AB, a representative gram-negative pathogen, and EF, a representative gram-positive pathogen.Both AB and EF are associated with different life-threatening infections.28,29Pyrimidine-defective mutant pyrB::tn was selected as a representative auxotrophic mutant for this experiment.Furthermore, we confirmed the pyrB::tn mutant to be truly deficient in pyrimidine biosynthesis using chemical complementation with uridine-5′-monophosphate (Supplemental Figure 1) as well as whole genome sequencing (data not shown).Like earlier mixed culture experiments with JE2, the fitness or PA14 was found to be unaffected in mixed culture with AB and EF compared to monoculture.Whereas, compared to monoculture, a significant (p < 0.0001) rescue in pyrB::tn cells has been found in the presence of both AB and FE (Figure 2A).That tells us that the growth rescue of the pyrimidine-defective mutants is not restricted to SA or gram-negative or gram-positive pathogens.Neighboring pathogens with functional pyrimidine biosynthesis machinery may rescue pyrimidine-defective mutants of PA in infection sites.Furthermore, both the wildtype PA14 and pyrB::tn mutant were found to be slightly competitive (p < 0.05) against AB, whereas EF was found to be unresponsive to PA14 and pyrB::tn mediated killing in mixed culture (Figure 2A).That tells that the molecules responsible for the pyrimidine complementation may come from metabolically active or lysed cells." Ln 182 -'less virulent' does not seem like an appropriate term here, since these experiments were all performed in the absence of host cells Response: Thanks so much for your feedback.We deleted 'virulent' from the statement, and now the statement would be "mutants may be attributed to the fact that the auxotrophic strains are less competitive against JE2 compared to PA14" in lines 251-252 Ln 183-184 -eDNA was not examined in Fig 1, so conclusions about its role in rescue of the PA14 pyrimidine auxotrophs are not justified Response: We appreciate your feedback.We agree with the point that it would be too early to claim eDNA as the rescuer.Based on reviewers comment, we revised the statements as follows in lines 240-245."These data demonstrate that JE2 can rescue the pyrimidine-deficient PA in mixed cultures.This rescue might be achieved through release of eDNA, nucleosides, or nitrogenous bases.The release of eDNA or other secretory molecules in the liquid cultures may be a result of cellular degradation following lysis and/or secretion by metabolically active bacterial cells.To identify the possible source of these molecules, either from lysis or secretion of metabolically active bacteria, we estimated the fitness of JE2 in mono and mixed cultures (Figure 1C)."Response: We started this experiment with similar cell density, but we did not normalize eDNA to CFU/ml for each culture.Our main goal of this experiment was to measure the secreted eDNA in the culture over time.We agree that the defective growth of the PA mutant would produce less eDNA.Actually, we wanted to explore this particular phenomenon with this experiment to show that these mutants are incapable of producing enough pyrimidine in monoculture, therefore creating less eDNA, which in turn makes them growth defective as pyrimidines are essential for their growth and development.
Ln 192-194 -Statistics for this comparison (JE2 and pyrimidine mutants) are not shown in the figure .Response: We appreciate your feedback.We have revised the figure 3 accordingly.
Ln 194-196 -It's not clear how JE2 having more eDNA than the PA mutants validates the PA mutants' auxotrophy.The conclusion about DNA secretion vs cell lysis is also not clear.Response: Thanks so much for your comment.We agree that, without further studies, it would not be justifiable to claim the PA mutant's auxotrophy just by comparing the eDNA in JE2 monoculture.Therefore, we revised the manuscript to address this concern and tried to make it clearer, as follows: lines 287-293."Our results depict that the presence of eDNA in JE2 monoculture is higher than in PA's pyrimidinedefective mutants (pyrB::tn; p>0.05, pyrD::tn; p<0.05, pyrE::tn; p<0.05, pyrF::tn; p>0.05) which might be attributed to the low growth of the defective mutants in monoculture (Figure 3).Different microorganisms, including bacteria, may release eDNA in the environment through various mechanisms, such as active secretion by metabolically active cells, by membrane vesicles, or following cell lysis," Ln 210-211 -These two figures show that co-culture with JE2 can rescue growth of PA14 pyrimidine auxotrophs and that eDNA can be detected to varying extents in these cultures.eDNA as the cause of the growth rescue has not been established.

Response:
We really appreciate you pointing out this critical issue.Based on the reviewer's comment we revisited our data and also based on the new additional data we agree with the reviewer's concern that eDNA would be a minor contributor in pyrimidine complementation.We have revised the whole manuscript accordingly including our concluding remarks in lines 380-387."Both metabolically active and lysed cells may facilitate the pyrimidine complementation in neighboring pathogens.Importantly, secreted eDNA is a minor contributor in pyrimidine complementation towards rescuing the pyrimidine-defective PA, but neighboring cells (SA) are more proficient for pyrimidine exchange via intricate cell-to-cell contact mechanism.Apart from eDNA, secreted nucleotides, such as UMP, a precursor for pyrimidine nucleotide, may also rescue pyrimidine defective mutants.The role of secreted nucleosides and nitrogenous bases in this complementation requires further investigation." In addition to eDNA, we conducted a new chemical complementation experiment using UMP, a precursor for the pyrimidine nucleotide and a product of de novo pyrimidine biosynthesis.We found that UMP can significantly rescue the growth of a pyrimidinedefective mutant of PA as well.That means, secretory nucleotides can also play a role in pyrimidine exchange.Please find the added data in lines 261-263 and Supplemental Figure 1A "Furthermore, we confirmed the pyrB::tn mutant to be truly deficient in pyrimidine biosynthesis using chemical complementation with uridine-5′-monophosphate (Supplemental Figure 1) as well as whole genome sequencing (data not shown)" Fig 3 and 4 -These data seem to argue against the conclusion that eDNA mediates rescue of the pyrimidine auxotrophs.Very small growth differences are observed with eDNA supplementation and growth is not restored to the level of WT.Response: We really appreciate you pointing out this critical issue.Based on the reviewer's comment we revisited our data and also based on the new additional data we agree with the reviewer's concern that eDNA would be a minor contributor in pyrimidine complementation.We have revised the whole manuscript accordingly including our concluding remarks in lines 380-387."Both metabolically active and lysed cells may facilitate the pyrimidine complementation in neighboring pathogens.Importantly, secreted eDNA is a minor contributor in pyrimidine complementation towards rescuing the pyrimidine-defective PA, but neighboring cells (SA) are more proficient for pyrimidine exchange via intricate cell-to-cell contact mechanism.Apart from eDNA, secreted nucleotides, such as UMP, a precursor for pyrimidine nucleotide, may also rescue pyrimidine defective mutants.The role of secreted nucleosides and nitrogenous bases in this complementation requires further investigation."does not indicate the difference between panels A and B. Without this information, it is impossible to interpret the results.Both panels show co-cultures between pyrB mutant and JE2, but they show different results.

Respond:
We appreciate your feedback.We have revised the figure legend accordingly.Please find the correction in revised figure 6.
Simplifying the abbreviations for S. aureus and P. aeruginosa by using just one abbreviation each (either SA/PA or the strain names JE2/PA14) would help the reader follow the manuscript.Respond: We appreciate your feedback.We have revised the manuscript according to the reviewer's comments.Throughout the manuscript, PA and SA were used to refer to P. aeruginosa and S. aureus.PA14 denotes wild-type PA and JE2 denotes wild-type JE2

Reviewer #2 (Comments for the Author):
In this manuscript, Al Mahmud et al., present evidence that pyrimidines produced by S. aureus (SA) can rescue P. aeruginosa (PA) that lacks the ability to perform pyrimidine synthesis.The authors use a series of PA mutants created using a transposon each lacking a unique gene in pyrimidine synthesis.They then show that, alone, these mutant strains survive, but do not grow, in minimal medium.However, complementation with SA or herring DNA returns growth to the mutants.The authors then show that there is a likely a spatial component to the exchange.Taken together, the manuscript adds to our understanding of how PA and SA interact.
Overall, the manuscript is well written.The figures and figure legends are very clear, which I really appreciate.However, I am concerned that that it more than just pyrimidines that are involved in the rescue.It could be an entirely different substrate that feeds into pyrimidine synthesis that is overproduced by SA.I think that to truly show that it is pyrimidines that are recuing PA when co-culture with SA, the authors need to perform some important control experiments.
Response: We appreciate your feedback.To address this concern, we conducted a new chemical complementation experiment using UMP, a precursor for the pyrimidine nucleotide and a product of de novo pyrimidine biosynthesis.We found that UMP can significantly rescue the growth of a pyrimidine-defective mutant of PA as well.That means, secretory nucleotides can also play a role in pyrimidine exchange.Please find the added data in lines 261-263 and Supplemental Figure 1 "Furthermore, we confirmed the pyrB::tn mutant to be truly deficient in pyrimidine biosynthesis using chemical complementation with uridine-5′-monophosphate (Supplemental Figure 1) as well as whole genome sequencing (data not shown)."

Specific points:
1) The introduction is really short and misses a lot of interactions that have been previously shown between these two pathogens, both antagonistic and mutualistic.These should be summarized.Furthermore, because this manuscript involves several genes and (presumably) nitrogenous base scavenging, a brief paragraph on purine/pyrimidine synthesis and salvage is warranted to put the mutants (pyrF, etc.) in the greater context of the pathways in which they are found.

Response:
We really appreciate your feedback, which we believe increased our lit review quality.We incorporated these important studies in lines 56-60, 62-70, and 81-91."For example, iron depletion in co-culture may increase the lysis of SA by PA through secreted 2-alkyl-4(1H)-quinolones.4 Bacterial co-culture on human bronchial epithelial cell monolayers showed PA drives SA metabolism from aerobic respiration to fermentation and eventually kills SA by secreting siderophores or 2-heptyl-4-hydroxyquinoline N-oxide (HQNO).5"For example, PA isolated from coinfected patients is found to be less competitive against SA; in fact, PA with mucoid phenotype would become severely inactive against SA and would reside within the infection site together.Alginate-producing mucoid strains of PA downregulate the synthesis of different virulence factors essential for the killing of SA.7 In the polymicrobial lung infection model, virulence factors secreted by SA are found to be helpful in the proliferation, spread, and pathogenicity of gram-negative pathogens like PA through compromising host immunity.8In addition, host immune proteins like calprotectin may facilitate the co-colonization of these two classical competitors, PA and SA, in cystic fibrosis lung.9 " "Pathogenic bacteria rely on de novo nucleotide biosynthesis to initiate infection, survive, and be virulent.Regulators of this process have been shown to be essential in regulating the production of virulence factors.14,15Synthesis or acquisition of purines and pyrimidines is essential for cellular functions and the reproduction of microorganisms.14For example, pyrimidine biosynthetic genes are essential for PA to grow well in the CF-infected lung environment.16In de novo purine biosynthesis, precursor molecule 5-phosphoribosyl-α-1-pyrophosphate (PRPP) is converted into the final product inosine-5′-monophosphate (IMP) by the action of different enzymes, encoded by genes such as purF, purD, purN, purT, purS, purQ, url, purM, purK, purE, purC, purB, and purH, etc.14 Similarly, two methods are employed by organisms for obtaining pyrimidines: de novo synthesis, which is a universal process consisting of six consecutive enzyme reactions, or a salvage pathway.17In contrast, in de novo pyrimidine biosynthesis, the precursor molecule carbamoyl phosphate (CP) is converted into the final product uridine-5′monophosphate (UMP) by the action of different enzymes, namely carbamyl phosphate synthetase, aspartate transcarbamylase, dihydroorotase, dihydroorotate dehydrogenase, orotate phosphoribosyltransferase, and orotidylate decarboxylase.18,19These enzymes are encoded by six unlinked genes, namely pyrB, purC, pyrK, pyrD, pyrE, and pyrF, etc.14 Finally, the precursor molecule UMP can be converted to different pyrimidines, such as UDP, UTP, CTP, etc., by the action of other enzymes.Apart from these, UMP can be produced from pyrimidine bases and nucleosides by enzymatic action through salvage pathway.17" 2) Throughout the first part of the results and discuss, the reader is made to think that eDNA is being secreted by and that is rescuing PA.But SA could also produce metabolites that feed into pyrimidine synthesis, which may be rescuing PA.If it was simply a matter of providing pyrimidines, then shouldn't all of the mutants be rescued more or less in the exact same way.
That is, in figure 3, shouldn't all of the mutants behave, for example, like pyrF?I understand that this data is not in the presence of SA but because cytosine enters pyrimidine synthesis after purF (and thus all of the other enzymes that they have knocked out) and thymine enters via a route that doesn't involve the pyr enzymes, shouldn't they all behave the same?This to me is very puzzling...If it was simply just pyrimidines, could the PA mutants be rescued with supplementation with pyrimidines?Have the authors tried these experiments (as opposed to herring DNA)?To me, that would provide more evidence that only pyrimidines are responsible for rescue.

Response:
We really appreciate you pointing out this critical issue.Based on the reviewer's comment we revisited our data and also based on the new additional data we agree with the reviewer's concern that eDNA would be a minor contributor in pyrimidine complementation.We have revised the whole manuscript accordingly including our concluding remarks in lines 380-387."Both metabolically active and lysed cells may facilitate the pyrimidine complementation in neighboring pathogens.Importantly, secreted eDNA is a minor contributor in pyrimidine complementation towards rescuing the pyrimidine-defective PA, but neighboring cells (SA) are more proficient for pyrimidine exchange via intricate cell-to-cell contact mechanism.Apart from eDNA, secreted nucleotides, such as UMP, a precursor for pyrimidine nucleotide, may also rescue pyrimidine defective mutants.The role of secreted nucleosides and nitrogenous bases in this complementation requires further investigation." In addition to eDNA, we conducted a new chemical complementation experiment using UMP, a precursor for the pyrimidine nucleotide and a product of de novo pyrimidine biosynthesis.We found that UMP can significantly rescue the growth of a pyrimidinedefective mutant of PA as well.
Please find the added data in lines 261-263 and Supplemental Figure 1 "Furthermore, we confirmed the pyrB::tn mutant to be truly deficient in pyrimidine biosynthesis using chemical complementation with uridine-5′-monophosphate (Supplemental Figure 1) as well as whole genome sequencing (data not shown)" We agree that it is interesting that we are seeing varying rescues between different mutants.Because of time limitations, we could not include all the pyrimidine-defective mutants for the extended experiments.However, we think it would be exciting to explore this phenomenon further in future studies.
3) Another way to provide support for pyrimidines in rescue in acquiring SA that lacks a gene in pyrimidine synthesis early on in the pathway (right after the prpp autoregulation edge in the pathway).Co-culture with this strain should not lead to growth of either strain.That would provide more conclusive evidence that it is pyrimidines (nitrogenous bases or the deoxy form) that is rescuing PA.
showed that JE2 supernatant does not affect the fitness of PA14, but it significantly (p < 0.05) increases the fitness of pyrB::tn cells (Figure 6C).Interestingly, similar to eDNA, the rescue of pyrimidinedefective mutants mediated by JE2 supernatant is only partial compared to the complete rescue observed in mixed culture with JE2.JE2 supernatant may contain eDNA, pyrimidine nucleotides, pyrimidine nucleosides, or nitrogenous bases that can complement the pyrimidine deficiencies in auxotrophic bacteria." Overall, I think that that authors have a really good idea, and if it is supported by additional, critical control experiments above, would certainly have an impact on the field.But without the experiments above, while it could be pyrimidines that are exchanged, it might also be a number of other things that are rescuing the PA mutant strains.
Fig 2 -Was eDNA quantification normalized to CFU/ml for each culture?Fig 1 shows that the pyrimidine mutants have a growth defect.Less dense cultures would be expected to contain less eDNA.Ln 192-194 -Statistics for this comparison (JE2 and pyrimidine mutants) are not shown in the figure.

Fig 2 -
Fig 2 -Was eDNA quantification normalized to CFU/ml for each culture?Fig 1 shows that the pyrimidine mutants have a growth defect.Less dense cultures would be expected to contain less eDNA.Response: We started this experiment with similar cell density, but we did not normalize eDNA to CFU/ml for each culture.Our main goal of this experiment was to measure the secreted eDNA in the culture over time.We agree that the defective growth of the PA mutant would produce less eDNA.Actually, we wanted to explore this particular phenomenon with this experiment to show that these mutants are incapable of producing enough pyrimidine in monoculture, therefore creating less eDNA, which in turn makes them growth defective as pyrimidines are essential for their growth and development.